Controlled-Released Peptide Formulations

ABSTRACT

Described herein are methods and compositions for modulating the release and/or drug loading characteristics of encapsulated bioactive agents in polymer-based delivery systems via direct modification of the isoelectric point and/or net charge of the bioactive agent.

FIELD

The invention relates to peptide formulations, and more specifically tomethods and compositions for modifying the release and/or drug-loadingcharacteristics of such formulations.

BACKGROUND OF THE INVENTION

The importance of biocompatible and/or biodegradable polymers ascarriers for active therapeutic agents is well established.Biocompatible, biodegradable, and relatively inert polymers such aspoly(lactide) (PL) or poly(lactide-co-glycolide) (PLGA) containing abioactive agent are commonly utilized in controlled-release deliverysystems (for review, see M. Chasin, Biodegradable polymers forcontrolled drug delivery. In: J. O. Hollinger Editor, BiomedicalApplications of Synthetic Biodegradable Polymers CRC, Boca Raton, Fla.(1995), pp. 1-15; T. Hayashi, Biodegradable polymers for biomedicaluses. Prog. Polym. Sci. 19 4 (1994), pp. 663-700; and Harjit Tamber, PalJohansen, Hans P. Merkle and Bruno Gander, Formulation aspects ofbiodegradable polymeric microspheres for antigen delivery, Advanced DrugDelivery Reviews, Volume 57, Issue 3, 10 Jan. 2005, Pages 357-376).

With respect to the delivery of therapeutic peptides in particular,however, there still exist many challenges to the design of effectivecontrolled-release, polymer-based delivery systems. A basic requirementfor such delivery systems is appropriate control over the release of theencapsulated active agent, an objective which is complicated byvariations in the release kinetics of polymer systems. Generally, aninitial diffusional or burst release phase from the intact polymersystem is followed by a slower lag phase leading to an erosional releasephase as the polymer system begins to degrade. It is important tomaintain the concentration of the peptide molecule within atherapeutically effective window throughout both of the principalpeptide release phases and to avoid excessive concentrations, andparticularly an initial burst during the diffusional release phase,which may lead to adverse side effects or untoward results. In thisrespect, however, wide variation in the size, charge and conformation ofdifferent peptide molecules has thus far prevented a more uniformapproach to their effective encapsulation.

The prior art describes various strategies for improvingcontrolled-release delivery from polymer-based delivery systemsincluding the use of new polymeric materials and polymer blends, and/orthe incorporation of additives in such systems to help facilitate drugrelease. U.S. Pat. No. 7,326,425, for example, describes a blendedpolymer-based delivery system having a first polymer capable of forminghydrogen bonds with a desired bioactive agent to prevent bursts, and asecond polymer the degradation products of which trigger the release ofthe active agent from the first polymer. Alternatively, U.S. PatentPublication No. 2007/0092574 describes the addition of certain organicions to polymer-based delivery systems encapsulating water-solublebioactive agents to reduce the burst release and degradation of thebioactive agent, wherein the organic ion is selected to neutralize theoverall charge of a particular bioactive agent.

In each of these examples, however, and in the prior art in general, theprimary focus of such strategies is on manipulation of the polymer-baseddelivery system to suit the requirements of a particular bioactiveagent, as opposed to manipulation or adaptation of the bioactive agentitself.

SUMMARY OF THE INVENTION

In contravention of the conventional formulation methodology ofmanipulating the polymer-based delivery system to suit the encapsulatedagent, the present invention provides methods and compositions formodulating the release and/or drug-loading characteristics of anencapsulated bioactive agent through direct modification of thebioactive agents themselves. As demonstrated herein, modification of theisoelectric point of a bioactive agent such as a peptide molecule, e.g.,alteration of the overall charge of the peptide, can predictably modifythe release and/or loading characteristics of polymer-based deliverysystems in clinically meaningful ways including, e.g., reducing orenhancing the initial diffusional release of the peptide from thepolymer-based delivery system, modulating the erosional release ratefrom biodegradable systems, and/or increasing the encapsulationefficiency of such peptides.

In one aspect, methods for increasing bioactive agent loading efficiencyin polymer-based delivery systems are provided, comprising modifying theisoelectric point of the agent prior to encapsulation in a polymer-baseddelivery system. In one embodiment, the isoelectric point of the agentis modified such that it carries a more positive charge compared to theparent molecule in the environment of the desired polymer-based deliverysystem.

In one embodiment, methods for increasing bioactive agent loadingefficiency in polymer-based delivery systems are provided, comprisingadding additional positive charge to a parent molecule.

In one aspect, methods for modulating the erosional release rate of abioactive agent from a biodegradable polymer-based delivery system areprovided, comprising changing the isoelectric point of the agent priorto encapsulation in the polymer-based delivery system. In oneembodiment, the isoelectric point of an agent is quantitativelyincreased or decreased such that it carries a greater net positive ornegative charge, respectively, compared to the parent molecule in theenvironment of the desired polymer-based delivery system.

In one embodiment, methods for increasing the erosional release rate ofa bioactive agent from a biodegradable polymer-based delivery system areprovided, comprising adding additional positive or negative charge to aparent molecule to produce a stoichiometric increase or decrease,respectively, in net charge relative to the parent molecule. In oneembodiment, additional positive charge is added to a neutral or cationicparent molecule to increase the erosional release rate. In anotherembodiment, additional negative charge is added to a neutral or anionicparent molecule to increase the erosional release rate. In a preferredembodiment, acid-terminated polymer-based delivery systems are employedfor enhanced effect.

Surprisingly, the present inventors have determined that an increase inthe net positive charge of a bioactive agent relative to a cationicparent molecule can work as well as, and in some cases even better than,a reduction in or neutralization of the net charge to increase theerosional release rate of such an agent from a biodegradablepolymer-based delivery system. Significantly, as also demonstrated forthe first time herein, creating a greater charge density in a chargedbioactive agent relative to a parent molecule provides for greatereffect.

In one aspect, methods for modulating the initial diffusional release ofa bioactive agent from a polymer-based delivery system are alsoprovided, comprising changing the isoelectric point of the agent priorto encapsulation in the polymer-based delivery system. In oneembodiment, the isoelectric point of the agent is increased or decreasedsuch that it carries a greater net positive or negative charge,respectively, relative to the parent molecule in the environment of thedesired polymer-based delivery system.

In one embodiment, methods for increasing the initial diffusionalrelease of a bioactive agent from a polymer-based delivery system areprovided, comprising adding additional positive charge to the parentmolecule to produce a stoichiometric increase in net charge relative tothe parent molecule. In a preferred embodiment, ester-terminatedpolymer-based delivery systems are employed for enhanced effect.

In one embodiment, methods for decreasing the initial diffusionalrelease of a bioactive agent from a polymer-based delivery system areprovided, comprising adding additional negative charge to the parentmolecule to produce a stoichiometric decrease in net charge relative tothe parent molecule. In a preferred embodiment, ester-terminatedpolymer-based delivery systems are employed for enhanced effect.

Any suitable means for modifying the isoelectric point of a bioactiveagent can be employed in conjunction with the subject methods including,e.g., chemical modification, amino acid substitution, conjugation ofpositively or negatively-charged accessory molecules, and morepreferably cleavable accessory molecules, and the like. The additionalpositive or negative charge may be distributed uniformly ornon-uniformly across the bioactive agent, and is preferably implementedat a location distal to the known site(s) of action of the parentmolecule, e.g. binding site, etc. In one embodiment, the additionalcharge distribution is clustered at the amino or carboxy terminus. Inanother embodiment, the additional charge is conjugated to an amino acidside chain.

Modification of the isoelectric point as disclosed herein may also beemployed in conjunction with more conventional techniques such asconversion to water insoluble addition salts (e.g., U.S. Pat. No.5,776,886), pegylation (U.S. Pat. No. 6,706,289), and the like, tofurther modulate release kinetics and/or loading efficiency. In afurther aspect, improved controlled-release pharmaceutical compositionsare provided comprising bioactive agents modified according to the abovemethods and encapsulated in polymer-based delivery systems.

In one embodiment, the controlled-release pharmaceutical compositioncomprises a modified bioactive agent encapsulated by a polymer, whereinthe isoelectric point of the modified bioactive agent has been increasedrelative to the parent molecule to increase drug loading efficiency,and/or to increase the erosional release rate of the modified bioactiveagent, preferably from an acid-terminated polymer system, and/or toincrease the diffusional release of the modified bioactive agent,preferably from an ester-terminated polymer system. In one suchembodiment, the parent molecule is neutral or cationic.

In another embodiment, the controlled-release pharmaceutical compositioncomprises a modified bioactive agent encapsulated by a biodegradablepolymer, wherein the isoelectric point of the modified bioactive agenthas been decreased relative to the parent molecule to increase theerosional release rate of the modified bioactive agent, preferably froman acid-terminated polymer system, and/or to decrease the diffusionalrelease rate of the modified bioactive agent, preferably from anester-terminated polymer system, relative to the parent molecule. In onesuch embodiment, the parent molecule is neutral or anionic.

Unless otherwise specified, the compositions described herein maycomprise a non-biodegradable polymer-based delivery system, e.g., apolymer system comprising a non-biodegradable polymer. In one aspect,the non-biodegradable polymer is selected from the group consisting ofpolyacrylates, polymers of ethylene-vinyl acetates and other acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blends andcopolymers thereof.

In another aspect, the compositions described herein may comprise abiodegradable polymer-based delivery system, e.g., a polymer systemcomprising a biodegradable polymer. In another aspect, the biodegradablepolymer is selected from the group consisting of homopolymers ofpoly(lactic acid) (PLA), polylactide (PL) or poly(glycolic acid) (PGA),polyglycolide (PG), poly(lactic acid)-co-(glycolic acid) (PLGA),poly(actide-co-glycolide (PLG), poly(ortho esters), and polyanhydrides.Due to the biocompatibility and the long history of their clinicalapplications, PLGA and PLA are most generally used. Other biodegradablepolymers that may be used include polycaprolactone, polycarbonates,polyesteramides, poly(amino acids), poly(dioxanones), poly(alkylenealkylate)s, polyacetals, polycyanoacrylates, biodegradablepolyurethanes, blends and copolymers thereof.

The subject compositions and methods find advantageous use with avariety of bioactive agents, including therapeutic proteins, nucleicacids, peptides, polypeptides, oligonucleotides, and the like.

In another aspect, the invention provides methods of treating a patientin need of treatment, comprising administering a therapeuticallyeffective amount of a pharmaceutical composition of the invention to thepatient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary schematic of a hypothesized triphasic drugrelease from a degradable matrix.

FIG. 2 shows the actual loading (y-axis) of five peptides (DP1, DP2,DP3, DP4, and DP5) with a neutral, positive (+) or negative (−) chargeoverall (x-axis). Unless otherwise noted, each peptide molecule wasloaded into the polymer as an acetate salt.

FIG. 3 shows the percent theoretical loading efficiency (y-axis) of fivepeptides (DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) ornegative (−) charge overall (x-axis). Unless otherwise noted, eachpeptide molecule was loaded into the polymer as an acetate salt.

FIG. 4 shows the mean particle size (μM) of five peptides (DP1, DP2,DP3, DP4, and DP5) with a neutral, positive (+) or negative (−) chargeoverall (x-axis). Unless otherwise noted, each peptide molecule wasloaded into the polymer as an acetate salt.

FIG. 5 shows the release rate as percent release (y-axis) of fivepeptides (DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) ornegative (−) charge overall over 35 days (x-axis) from a microparticle,polymer-based formulation containing an acid-terminated, 50:50poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Unlessotherwise noted, each peptide molecule was loaded into the polymer as anacetate salt.

FIG. 6 shows the release rate as percent release (y-axis) of fivepeptides (DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) ornegative (−) charge overall over 14 days (x-axis) from a microparticle,polymer-based formulation containing an acid-terminated, 50:50poly(lactide-co-glycolide) with an approximate intrinsic viscosity(i.v.) of 0.2 dL/g as measured in chloroform at a concentration of 0.5g/dL at 30° C. Unless otherwise noted, each peptide molecule was loadedinto the polymer as an acetate salt.

FIG. 7 shows the release rate as percent release (y-axis) of fourpeptides (DP1, DP2, DP3, and DP5) with a neutral, positive (+) ornegative (−) charge overall over 17 days (x-axis) from a microparticle,polymer-based formulation containing an ester-terminated, 50:50poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Unlessotherwise noted, each peptide molecule was loaded into the polymer as anacetate salt.

FIG. 8 shows the release rate as percent release (y-axis) of fivepeptides (DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) ornegative (−) charge overall over 29 days (x-axis) from a microparticle,polymer-based formulation containing an acid terminated 85:15poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Unlessotherwise noted, each peptide molecule was loaded into the polymer as anacetate salt.

FIG. 9 shows the release rate as percent release (y-axis) of fivepeptides (DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) ornegative (−) charge overall over 65 days (x-axis) from a microparticle,polymer-based formulation containing an ester-terminated 85:15poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Unlessotherwise noted, each peptide molecule was loaded into the polymer as anacetate salt.

FIG. 10 shows the release rate as percent release (y-axis) of fivepeptides (DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) ornegative (−) charge overall over 15 days (x-axis) from a microparticle,polymer-based formulation containing an ester-terminated 85:15poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Unlessotherwise noted, each peptide molecule was loaded into the polymer as anacetate salt.

DETAILED DESCRIPTION

Methods and formulations are provided for the controlled-release ofbioactive agents from polymer-based delivery systems through directmodification of the bioactive agent. As described herein, theisoelectric point of the modified bioactive agent may be changedrelative to a parent molecule, and/or the net charge of the modifiedbioactive agent may be changed relative to a parent molecule, etc.Encapsulated formulations comprising such modified bioactive agents haveenhanced controlled-release properties, e.g., a lower initialdiffusional or burst release, an increased erosional release rate,increased encapsulation efficiency, etc., compared to formulationscomprising similarly encapsulated formulations of the parent molecule.

Release of a bioactive agent from a polymer, e.g., a biodegradablepolymer such as a PLG microparticle, generally follows a triphasicrelease profile as exemplified in FIG. 1. Phase 1 may generally becharacterized as a diffusional release or “burst” effect, during whichthe initial release rate of the modified peptide molecule may be rapid,and may be dependent on hydration of the polymer (occurring withinminutes), swelling of the matrix (hours), dissolution of the modifiedpeptide molecule (minutes) and diffusion from the matrix (hours).

The second phase of release (Phase 2, FIG. 1) may be referred to as theinduction or lag phase, and may be characterized by a period of sloweror no release. Phase 2 may be associated with the time required forpores or channels to form or time for water to fill such pores orchannels in the polymer matrix thereby allowing access to the modifiedpeptide molecule entrapped within the polymer matrix.

When a biodegradable polymer-based delivery system is used, and asbiodegradable polymer degradation continues, diffusional paths may beformed through the eroding matrix, which may allow the modified peptidemolecule to travel down a concentration gradient and escape the matrix.This erosional release corresponds to the third phase of release asdemonstrated in FIG. 1.

Release of a bioactive agent from a non-biodegradable polymer generallyfollows a biphasic release profile, in which phase 1 corresponds to adiffusional release of the bioactive peptide and phase 2 corresponds toa lag phase. Accordingly, skilled artisans are generally familiar withtypical release rates of bioactive agents from such polymer-baseddelivery systems.

In one embodiment, improved controlled-release compositions and methodsare provided wherein the isoelectric point of a parent molecule isincreased to produce a modified bioactive agent having a more positivenet charge and/or charge density, which as demonstrated herein canincrease drug-loading efficiency, increase the erosional release rate ofthe modified bioactive agent, and particularly from acid-terminatedpolymer-based systems, and/or increase the diffusional release of themodified bioactive agent, and particularly from ester-terminatedpolymer-based systems, relative to the parent molecule. In one suchembodiment, the parent molecule is neutral or cationic.

In another embodiment, improved controlled-release compositions andmethods are provided wherein the isoelectric point of a parent moleculeis decreased to produce a modified bioactive agent having a morenegative net charge and/or charge density, which as demonstrated hereincan increase the erosional release rate of the modified bioactive agent,and particularly from acid-terminated polymer-based systems, and/ordecrease the diffusional release rate of the modified bioactive agent,and particularly from ester-terminated polymer-based systems, relativeto the parent molecule. In one such embodiment, the parent molecule isneutral or anionic.

“Bioactive agent” as used herein refers to any therapeutic protein,therapeutic antibody, nucleic acid, peptide, polypeptide,oligonucleotide, aptamer or other biologically active compound foradministration to a subject.

By “peptide molecule” as used herein is meant a polymeric moleculecomprising at least two amino acids covalently linked by a peptide bond,and includes a protein, a polypeptide, an oligopeptide and a peptide. Apeptide molecule may be made up of naturally occurring amino acids andpeptide bonds, or synthetic peptidomimetic structures, i.e., “analogs”,such as peptoids (see Simon et al., 1992, Proc Natl Acad Sci USA89(20):9367, incorporated by reference).

By “amino acid” and “amino acid identity” as used herein is meant one ofthe twenty naturally occurring amino acids or any non-natural analoguesthat may be present at a specific, defined position. Thus “amino acid”,or “peptide residue”, as used herein means both naturally occurring andsynthetic amino acids (including analogues of naturally occurring aminoacids). For example, homophenylalanine, citrulline,2-amino-3-guanidinoproprionic acid, 2-amino-3-ureidoproprionic acid,Lys(Me), Lys(Me)₂, Lys(Me)₃, Ornitine, Omega-nitro-arginine, Arg(Me)2,α-methyl Arg, α-methyl Lys, homolysine, homoarginine, noreleucine, andthe like are considered amino acids for the purposes of the invention.“Amino acid” also includes imino acid residues such as proline andhydroxyproline. The side chain may be in either the (R) or the (S)configuration, and may be either D- or L-isomers. In the preferredembodiment, the amino acids are in the (S) or L-configuration, althoughD-isomers may be used to improve serum stability. If non-naturallyoccurring side chains are used, non-amino acid substituents may be used,for example to prevent or retard in vivo degradation.

“Parent molecule” as used herein refers to a bioactive agent that issubsequently modified in accordance with the invention teachings togenerate a “modified bioactive agent.” In some embodiments, a parentmolecule may be any bioactive agent molecule requiring acontrolled-release, polymer-based formulation for therapeutic use. Asdescribed herein, encapsulation and release from polymers can bemanipulated by modification of the parent molecule, e.g., modificationof the isoelectric point, net charge, solubility etc. of the parentmolecule.

Similarly, by “parent peptide molecule,” “parent polypeptide,” “parentprotein,” and the like as used herein is meant a polypeptide, proteinand the like that is subsequently modified to generate a “modifiedpeptide molecule.” For example, a parent peptide molecule, a parentpolypeptide, a parent protein or the like may serve as a template and/orbasis for at least one modification described herein, e.g., modificationof the isoelectric point, modification of the net charge, modificationof the solubility, etc. Said parent peptide molecule may be a naturallyoccurring polypeptide, or a variant or engineered version of a naturallyoccurring polypeptide. Parent polypeptide may refer to the polypeptideitself, compositions that comprise the parent polypeptide, or the aminoacid sequence that encodes it.

By “isolectric point”, “pI”, or the like as used herein is meant the pHat which a peptide molecule carries no net electrical charge. Theisoelectric point may be determined using well known methods, e.g., byisoelectric focusing. In case of smaller peptide molecules theapproximate pI may be also calculated. In general, the pI of a peptidemolecule depends on the pKa values of its basic and acidic groups, e.g.,in case of a peptide formed with encoded amino acids only, the primaryamine of the N-terminus or the lysine side chain, the guanidine group ofthe arginine side chain, the imidazole ring system of histidine and thecarboxylic acid groups of the peptide C-terminus, the aspartic acid sidechain and glutamic acid side chain.

Modification of the pI of a parent peptide molecule may occur by, e.g.,chemical modification. Non-limiting examples of such modificationsinclude acylation, alkylation or other chemical modification of theN-terminal amine group; amidation, esterification or other chemicalmodification of the C-terminal carboxylic acid group; substitution of anon-ionizable amino acid residue for a lysine, histidine, arginine,glutamic acid, aspartic acid or other non-encoded amino acids withacidic or basic chain groups; acylation, alkylation or other chemicalmodification of side chain groups of lysine, histidine, arginine orbasic functions of other, non-encoded amino acids; amidation,esterification or other chemical modification of side chain carboxylicacid groups, conjugation with pI shifting accessory molecules. etc. Incase of ionized peptides the pI of the salt form also depends on thepK_(a) of the counter ion.

As used herein, the “net charge” of a parent peptide molecule depends onits pI and the pH of the peptide solution. The net charge of a parentpeptide molecule may be modified by any of the following non-limitingexamples: acylation, alkylation or other chemical modification of theN-terminal amine group; amidation, esterification or other chemicalmodification of the C-terminal carboxylic acid group; substitution of anon-ionizable amino acid residue for a lysine, histidine, arginine,glutamic acid, aspartic acid or other non-encoded amino acid with acidicor basic chain groups; modification of the isoelectric point of theparent peptide; conjugation with positively or negatively chargedaccessory molecules, and the like.

As disclosed herein, modification of charge distribution and/or densitycan also be considered for modulation of polymer encapsulation andrelease properties of the parent peptide. Added charge density may bedistributed uniformly or non-uniformly across the modified peptidemolecule. In one embodiment, a non-uniform distribution of additionalnegative or positive charge comprises clustering the additional negativeor positive charge, respectively, at one or more positions within thepeptide chain. The cluster(s) of additional negative or positive chargemay be anywhere along the peptide, e.g., near or at the end of thepeptide, near or at the center of the peptide, etc., but are preferablypositioned distal to the active site of the peptide, which can bereadily determined by reference to the known characteristics of theparent molecule. Alternatively, the additional negative or positivecharge may be distributed evenly along the polymer.

In one embodiment, a modified peptide molecule may comprise anadditional negative or positive charge relative to its parent peptidemolecule, e.g., via substitution of appropriate amino acids. In oneembodiment, the addition of positive charge is accomplished bysubstitution of negative and/or non-ionizable amino acids in the parentpeptide molecule with lysine, arginine, histidine, or analogues thereof.In another embodiment, the addition of negative charge is accomplishedby substitution of positive and/or non-ionizable amino acids in theparent peptide molecule with glycine, aspartic acid, glutamic acid, oranalogues thereof (e.g., any alpha or beta amino alkanedioic acid (e.g.,amino suberic acid).

In one embodiment, a modified peptide molecule may comprise anadditional negative or positive charge relative to its parent peptidemolecule, e.g., via conjugation with one or more negatively-chargedaccessory molecule(s) or positively-charged accessory molecule(s),respectively. “Conjugation” as used herein refers to covalent linkage oftwo molecules as opposed to mere complexation or other close physicalassociation. Exemplary negatively-charged accessory molecules includeconjugations of in general any chemical functionality of a peptide suchas the hydroxyl group of tyrosine, threonine and serine side chains, thethiol group of the cysteine side chain or the N-terminal amino group oramino groups of the arginine and lysine side chains with phospholipids(phosphoamid bound), mono-, di-, and/or tri-phosphates, sulphates,citrates, tartaric acids, polyaspartic, polyglutamic and diacids (e.g.oxalic acids, malonic acids, succinic acids, etc.). Exemplarynegatively-charged structures also include, but are not limited to,poly(glutamic acid), anionic lipids, poly(aspartic acid), and alginates.In some cases the chemical functionality of the peptide may also have tobe induced or introduced in order to facilitate conjugation (e.g.formation of reactive thioesters, aldhydes, hydroxylamines,alkylbromides, maleimides, etc). Exemplary positively-charged accessorymolecules include, polylysine, polyarginine, polyhistidine, poly(allylamine), poly(ethyl imine), chitosan or positively charged lipidstructures.

Accessory molecules may also comprise a “tail” of positive or negativeamino acids, and may be conjugated to the bioactive agent by a moreneutral linker moiety, e.g., polyethylene glycol (PEG), poly —CH₂—, andthe like.

Modified peptide molecules may further include pharmaceuticallyacceptable counterions, representative examples of which are set forthin Table 1 below.

TABLE 1 Potential counterions Salt Class Examples inorganic acidshydrochloride, sulfate, nitrate, phosphate sulfonic acids tosylate,mesylate, esylate, isethionate carboxylic acids acetate, proprionate,maleate, benzoate, salicylate, fumarate hydroxyacids citrate, lactate,succinate, tartrate, glycollate anionic amino acids glutamate, aspartatefatty acids stearate, hexanoate, octanoate, decanoate, oleate

Modification of solubility in water and/or organic solvents as well asalteration of the hydrophilicity of a parent peptide molecule may alsobe employed in conjunction with the subject methods to further modulatethe encapsulation and release characteristics of a peptide in apolymer-based delivery system. The solubility and/or hydrophilicity of apeptide therapeutic may be modified by changing its salt form or bypegylation as described in, e.g., U.S. Pat. No. 5,776,885 and U.S. Pat.No. 6,706,289, the disclosures of which are both expressly incorporatedby reference herein.

As used herein “relative to a parent peptide molecule” refers to achange (e.g., an increase or decrease) in a quantifiable characteristic,e.g., isoelectric point, net charge, etc., by a modified peptidecompared to the parent peptide molecule (e.g., the peptide molecule thatwas subsequently modified to generate the modified peptide molecule)when the amounts of modified peptide molecule and parent peptidemolecule are essentially the same in the same assay.

Described herein are methods and compositions for modulating the releaseand/or drug loading characteristics of encapsulated peptide molecules inpolymer-based delivery systems through direct modification of thepeptide molecules themselves. Polymer-based delivery systems describedherein are generally biocompatible polymer-based delivery system. Thepolymer-based delivery systems described herein may comprise abiodegradable or non-biodegradable polymer, blends or copolymersthereof. A polymer-based delivery system, or a polymer, is biocompatibleif the polymer, and any degradation products of the polymer, arenon-toxic to the recipient and also present no significant deleteriousor untoward effects on the recipient's body.

Biocompatible, non-biodegradable polymers suitable for encapsulatingbioactive agents (e.g., peptide molecules) include, but are not limitedto, non-biodegradable polymers selected from the group consisting ofpolyacrylates, polymers of ethylene-vinyl acetates and other acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blends andcopolymers thereof.

Biodegradable polymers may also be used for encapsulating bioactiveagents (e.g., peptide molecules) e.g., for controlled-releaseformulations. In one embodiment, biodegradable polymers for which thedegradation products are known to be innocuous or biocompatible areused. Accordingly, such biodegradable polymers need not be surgicallyremoved at the end of a treatment. Commonly used biodegradable polymers,which have been investigated for the controlled-release of peptidemolecules, include homopolymers of poly(lactic acid) (PLA), polylactide(PL) or poly(glycolic acid) (PGA), polyglycolide (PG), poly(lacticacid)-co-(glycolic acid) (PLGA), poly(actide-co-glycolide (PLG),poly(ortho esters), and polyanhydrides. Due to the biocompatibility andthe long history of clinical applications, PLG and PL are most generallyused. Other biodegradable polymers that may be used includepolycaprolactone, polycarbonates, polyesteramides, poly(amino acids),poly(dioxanones), poly(alkylene alkylate)s, polyacetals,polycyanoacrylates, biodegradable polyurethanes, blends and copolymersthereof.

In one aspect, polymeric delivery systems can be microparticlesincluding, but not limited to microspheres, microcapsules, nanospheresand nanoparticles comprising biodegradable polymeric excipients,non-biodegradable polymeric excipients, or mixtures of polymericexcipients thereof, or the polymeric delivery systems can be, but notlimited to rods or other various shaped implants, wafers, fibers, films,in situ forming boluses and the like comprising biodegradable polymericexcipients, non-biodegradable polymeric excipients, or mixtures thereof.These systems can be made from a single polymeric excipient or a mixtureor blend of two or more polymeric excipients.

The term “microparticle” is used herein to include nanoparticles,microspheres, nanospheres, microcapsules, nanocapsules, and particles,in general. As such, the term microparticle refers to particles having avariety of internal structure and organizations including homogeneousmatrices such as microspheres (and nanospheres) or heterogeneouscore-shell matrices (such as microcapsules (and nanocapsules), porousparticles, multi-layer particles, among others. Microparticles areparticles that have sizes in the range of about 10 nanometers to about1000 micrometers (microns).

A variety of techniques known in the art can be used to formmicroparticles including e.g., single or double emulsion steps followedby solvent removal. Solvent removal may be accomplished by extraction,evaporation or spray drying among other methods.

In the solvent extraction method, the polymer is dissolved in an organicsolvent that is at least partially soluble in the extraction solventsuch as water. The modified bioactive agent, either in soluble form ordispersed as fine particles, is then added to the polymer solution, andthe mixture is dispersed into an aqueous phase that contains asurface-active agent such as poly(vinyl alcohol). The resulting emulsionis added to a larger volume of water where the organic solvent isremoved from the polymer/bioactive agent to form hardenedmicroparticles.

In the solvent-evaporation method, the polymer is dissolved in avolatile organic solvent. The bioactive agent, either in soluble form ordispersed as fine particles, is then added to the polymer solution, andthe mixture is suspended in an aqueous phase that contains asurface-active agent such as poly(vinyl alcohol). The resulting emulsionis stirred until most of the organic solvent evaporates, leavinginternally, solid microparticles.

In the spray drying method, the polymer is dissolved in a suitablesolvent, such as methylene chloride (e.g., 0.04 g/mL). A known amount ofthe modified bioactive agent is then suspended (if insoluble) orco-dissolved (if soluble) in the polymer solution. The solution or thedispersion is then spray dried. Microparticles ranging in diameterbetween one and ten microns can be obtained with a morphology, whichdepends on the selection of polymer.

Other known methods, such as phase separation and coacervation, andvariations of the above, are known in the art and also may be employedin the present invention.

A suitable polymeric excipient includes, but is not limited to, apoly(diene) such as poly(butadiene) and the like; a poly(alkene) such aspolyethylene, polypropylene, and the like; a poly(acrylic) such aspoly(acrylic acid) and the like; a poly(methacrylic) such as poly(methylmethacrylate), a poly(hydroxyethyl methacrylate), and the like; apoly(vinyl ether); a poly(vinyl alcohol); a poly(vinyl ketone); apoly(vinyl halide) such as poly(vinyl chloride) and the like; apoly(vinyl nitrile), a poly(vinyl ester) such as poly(vinyl acetate) andthe like; a poly(vinyl pyridine) such as poly(2-vinyl pyridine),poly(5-methyl-2-vinyl pyridine) and the like; a poly(styrene); apoly(carbonate); a poly(ester); a poly(orthoester) including acopolymer; a poly(esteramide); a poly(anhydride); a poly(urethane); apoly(amide); a cellulose ether such as methyl cellulose, hydroxyethylcellulose, hydroxypropyl methyl cellulose, and the like; a celluloseester such as cellulose acetate, cellulose acetate phthalate, celluloseacetate butyrate, and the like; a poly(saccharide), a protein, gelatin,starch, gum, a resin, and the like. These materials may be used alone,as physical mixtures (blends), or as co-polymers. Derivatives of any ofthe polymers listed above are also contemplated.

In one aspect, the polymeric excipient of the delivery system includes abiocompatible, non-biodegradable polymer such as, for example, asilicone, a polyacrylate; a polymer of ethylene-vinyl acetate; an acylsubstituted cellulose acetate; a non-degradable polyurethane; apolystyrene; a polyvinyl chloride; a polyvinyl fluoride; a poly(vinylimidazole); a chlorosulphonate polyolefin; a polyethylene oxide; or ablend or copolymer thereof.

In another aspect, the polymeric excipient includes a biocompatible,biodegradable polymer such as, for example, a poly(lactide); apoly(glycolide); a poly(lactide-co-glycolide); a poly(lactic acid); apoly(glycolic acid); a poly(lactic acid-co-glycolic acid); apoly(caprolactone); a poly(orthoester); a poly(phosphazene); apoly(hydroxybutyrate) or a copolymer containing a poly(hydroxybutarate);a poly(lactide-co-caprolactone); a polycarbonate; a polyesteramide; apolyanhydride; a poly(dioxanone); a poly(alkylene alkylate); a copolymerof polyethylene glycol and a polyorthoester; a biodegradablepolyurethane; a poly(amino acid); a polyetherester; a polyacetal; apolycyanoacrylate; a poly(oxyethylene)/poly(oxypropylene) copolymer, ora blend or copolymer thereof.

In one aspect, the polymer-based delivery system comprises anon-biodegradable polymer. In another aspect, the polymer-based deliverysystem comprises a biodegradable polymer, wherein the peptide isimbedded within the polymer of the delivery system. In one aspect, thepeptide is encapsulated in a delivery system composed ofpoly(lactide-co-glycolide), poly(lactide), poly(glycolide),polycaprolactone, poly(lactide-co-caprolactone),poly(lactide-co-glycolide-co-caprolactone),poly(glycolide-co-caprolactone), or a mixture thereof. Lactide/glycolidepolymers for drug-delivery formulations are typically made by meltpolymerization through the ring opening of lactide and glycolidemonomers. Some polymers are available with or without carboxylic acidend groups. Some polymers are available with a block of polyethyleneglycol (PEG). When the end group of the poly(lactide-co-glycolide),poly(lactide), or poly(glycolide) is not a carboxylic acid, for example,an ester, then the resultant polymer is referred to herein as blocked orcapped. The unblocked polymer, conversely, has a terminal carboxylicgroup.

In one aspect, linear lactide/glycolide polymers are used; howeverbranched polymers can be used as well. In certain aspects,high-molecular-weight polymers (e.g., i.v. >1 dL/g as measured inchloroform at a concentration of 0.5 g/dL at 30° C.) can be used formedical devices, for example, to meet strength requirements. In otheraspects, low-molecular weight polymers (e.g., i.v. <1 dL/g as measuredin chloroform at a concentration of 0.5 g/dL at 30° C.) can be used fordrug-delivery and vaccine delivery products where resorption time andnot material strength is more important. The lactide portion of thepolymer has an asymmetric carbon. Commercially racemic DL-, L-, andD-polymers are available. The L-polymers are more crystalline and resorbslower than DL-polymers. In addition to copolymers comprising glycolideand DL-lactide or L-lactide, copolymers of L-lactide and DL-lactide areavailable. Additionally, homopolymers of lactide or glycolide areavailable. Also, the lactide monomer can also contain alkyl groups.

In the case when the biodegradable polymer ispoly(lactide-co-glycolide), poly(lactide), or poly(glycolide), theamount of lactide and glycolide in the polymer can vary. In one aspect,the biodegradable polymer contains 0 to 100 mole %, 40 to 100 mole %, 50to 100 mole %, 60 to 100 mole %, 70 to 100 mole %, or 80 to 100 mole %lactide and from 0 to 100 mole %, 0 to 60 mole %, 10 to 40 mole %, 20 to40 mole %, or 30 to 40 mole % glycolide, wherein the amount of lactideand glycolide is 100 mole %. In one aspect, the biodegradable polymercan be poly(lactide), 85:15 poly(lactide-co-glycolide), 75:25poly(lactide-co-glycolide), or 65:35 poly(lactide-co-glycolide) wherethe ratios are mole ratios.

In one aspect, when the biodegradable polymer ispoly(lactide-co-glycolide), poly(lactide), or poly(glycolide), thepolymer has an intrinsic viscosity of from 0.15 to 1.5 dL/g, 0.25 to 1.5dL/g, 0.25 to 1.0 dL/g, 0.25 to 0.8 dL/g, 0.25 to 0.6 dL/g, or 0.25 to0.4 dL/g as measured in chloroform at a concentration of 0.5 g/dL at 30°C.

Pharmaceutical Compositions

In a further embodiment of the present invention, the modified peptidesand polymer-based delivery systems according to the subject inventionare admixed with an appropriate pharmaceutical carrier suitable foradministration in primates, especially humans, to provide pharmaceuticalcompositions.

Pharmaceutically acceptable carriers which can be employed in thepresent pharmaceutical compositions can be any and all solvents,dispersion media, isotonic agents and the like. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of the polymer-baseddelivery system or therapeutic peptide or other bioactive agentencapsulated therein, its use in the pharmaceutical compositions of thepresent invention is appropriate.

The carrier can be liquid, semi-solid, e.g., pastes, or solid carriers.Examples of carriers include oils, water, saline solutions, alcohol,sugar, gel, lipids, liposomes, resins, porous matrices, binders,fillers, coatings, preservatives and the like, or combinations thereof.

In a further embodiment of the present invention, the modified peptidesand polymer-based delivery systems according to the subject inventioncan be administered as a powder without carrier.

Methods of Treatment

In a further aspect of the present invention, methods are provided fortreating a disease in a vertebrate, preferably a mammal, preferably aprimate, with human subjects being an especially preferred embodiment,by administering a peptide formulation of the invention desirable fortreating such disease.

Experimental

EXAMPLE 1 Formulation of Model Peptides

Manufacturing Process:

The peptide-loaded microparticles were prepared using a standard solventextraction method. Approximately 200 mg of peptide was dissolved in 2grams of DMSO. Separately, 2 g of poly(lactide-co-glycolide (PLG) wasdissolved in 10 g of dichloromethane. The peptide solution was thenadded to the polymer solution while homogenizing at high revolutions perminute (rpm) using an IKA homogenizer. The peptide/polymer phase wasnext dispersed into a continuous phase consisting of 3 g of poly(vinylalcohol) (PVA) and 2.7 g of dichloromethane in 150 mL distilled water byhomogenizing at 700 rpm using a Silverson L4RT mixing assembly. Once thedroplets had been sufficiently formed (3 minutes) the emulsion wasdiluted with 1 L of distilled water and stirred on a stirplate for 1hour, allowing the extraction of the dichloromethane and solidificationof the PLG microparticles. Thereafter, the microparticles were isolatedby passing the suspension through a 125 micron sieve and collectingmicroparticles on a 20-micron sieve. The collected microparticles werethen lyophilized to remove residual water. The finished product was awhite to off-white free-flowing powder and was subsequently stored at−20° C.

Drug Content:

The drug content was determined by carefully extracting peptide from thePLG microparticle formulations. Typically, PLG formulations aredissolved in an appropriate organic solvent (or the polymer ishydrolyzed) and the drug is extracted into a suitable aqueous buffer.Drug in the extract is then quantified by HPLC. The concentration valueis used to determine the amount of drug contained in the microparticle,which is reported as weight percent (wt %).

In Vitro Release:

In-vitro release analysis consisted of placing samples of themicroparticle formulations into an appropriate receiving fluid (PBS atpH 7.4) maintained at 37° C. with mild agitation. The pH of thereceiving fluid was checked routinely to assure that the PBS maintains apH of 7.4. The receiving fluid was exchanged at various time points andthe amount of peptide released into the receiving fluid was quantifiedby HPLC. Control studies were performed to ensure the stability of thepeptide in the receiving fluid once released.

Microparticle Size

The mean size and size distribution of the microparticle formulationswas determined using a Beckman Coulter LS 13 320 Laser DiffractionParticle Size Analyzer.

FIGS. 2 and 3 show the effect of charge on drug load and loadingefficiency. Within the variation of the data, the drug load and loadingefficiency were consistent and similar across the neutral and positivelycharged peptides. The negatively charged peptide (DP5) was incorporated(loaded) less efficiently when compared to the other peptides studied.

FIG. 4 shows that, with the exception of DP2 in the microparticleformulation containing an ester-terminated 50:50poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C.,particle size was unaffected by the peptide incorporated.

FIGS. 5 and 6 demonstrate the effect of peptide charge on release from amicroparticle formulation containing an acid-terminated 50:50poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Thecharged peptides release at a consistently faster rate when compared tothe neutral peptide. It may also be noted that the higher charge-densitypeptides (DP3 DP4, DP5 verses DP2) released more rapidly. It is also ofnote that no “burst” was observed form this polymer-based deliverysystem.

FIG. 7 shows the release profiles form a similar microparticle,polymer-based formulation, an ester-terminated 50:50poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Whilethe neutral and negatively charged peptide showed very little releaseover the period studied, the positively charged peptide exhibited asignificant burst from the formulation. Further, the greater thepositive charge (higher charge density) the more significant the effecton the release rate.

FIG. 8 shows the release of the peptides from a microparticleformulation containing an acid-terminated 85:15poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Over theperiod studied, release of peptide was not observed from anymicroparticle formulation.

FIG. 9 shows the release of the peptides from a microparticleformulation containing an ester-terminated 85:15poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g asmeasured in chloroform at a concentration of 0.5 g/dL at 30° C. Whilethe neutral and negatively charged peptide showed very little releaseover the period studied, the positively charged peptide exhibited a moresignificant release from the microparticle formulation. The greater thepositive charge (higher charge density) the more significant the effecton the release rate.

EXAMPLE 2 Exemplary Modifications of Parent Peptides

Calcitonin

Calcitonin is a hormone used in the treatment of osteoporosis. The aminoacid sequence of human Calcitonin isCys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro(set forth as SEQ ID NO:1).

The pI of Calcitonin is modified to enhance its use in a controlledreleased formulation by adding a tri-lysine moiety at the N-terminus toincrease its encapsulation efficacy (set forth as SEQ ID NO:2).Alternatively, to increase its initial burst from an ester-terminatedpolyester, the glycine residues are replaced with lysines (set forth asSEQ ID NO:3). To increase erosional release from an acid-terminatedpolyester, the glycine residues are replaced with aspartic acids (setforth as SEQ ID NO:4).

Leuprolide

Leuprolide is a gonadotropin-releasing hormone agonist that may be usedin the treatment of prostate cancer or endometriosis. The amino acidsequence of Leuprolide is Glu-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NHEt (setforth as SEQ ID NO:5).

The pI of Leuprolide is modified to enhance its use in a controlledreleased formulation by replacing the glutamic acid with glutamine (setforth as SEQ ID NO:6) to increase its diffusional release from anester-terminated polyester and/or replacing the arginine with asparticacid (set forth as SEQ ID NO:7) to decrease diffusional release from anester-terminated polyester.

Octreotide

Octreotide is an octapeptide that may be used as an inhibitor of growthhormone and/or the treatment of acromegaly. The amino acid sequence ofOctreotide is DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr (set forth as SEQ IDNO:8).

The threonine is replaced with lysine (set forth as SEQ ID NO:9) tomodify the pI of Octreotide and to enhance its use in acontrolled-released formulation, e.g., to increase the encapsulationefficiency and drug load of Octreotide.

All citations are expressly incorporated herein in their entirety byreference.

We claim:
 1. A controlled-release pharmaceutical formulation comprisinga modified bioactive agent encapsulated by a polymer, wherein theisoelectric point of said modified bioactive agent has been increasedrelative to a parent molecule to increase drug loading efficiency. 2.The controlled-release pharmaceutical formulation according to claim 1,wherein the modified bioactive agent comprises additional positivecharge relative to a parent molecule.
 3. The controlled-releasepharmaceutical formulation according to claim 2, wherein said modifiedbioactive agent is conjugated to a positively-charged accessorymolecule.
 4. The controlled-release pharmaceutical formulation accordingto claim 2, wherein said modified bioactive agent comprisespositively-charged amino acid substitutions.
 5. The controlled-releasepharmaceutical formulation according to claim 3, wherein said modifiedbioactive agent is a peptide having the amino acid sequence set forth asSEQ ID NO:2.
 6. The controlled-release pharmaceutical formulationaccording to claim 4, wherein said modified bioactive agent is a peptidehaving the amino acid sequence set forth as SEQ ID NO:9.
 7. Acontrolled-release pharmaceutical formulation comprising a modifiedbioactive agent encapsulated by a biodegradable polymer, wherein theisoelectric point of said modified bioactive agent has been increased ordecreased relative to a parent molecule to increase the erosionalrelease rate.
 8. The controlled-release pharmaceutical formulationaccording to claim 7, wherein the modified bioactive agent comprisesadditional positive charge relative to a parent molecule.
 9. Thecontrolled-release pharmaceutical formulation according to claim 8wherein said modified bioactive agent is conjugated to apositively-charged accessory molecule.
 10. The controlled-releasepharmaceutical formulation according to claim 8, wherein said modifiedbioactive agent comprises positively-charged amino acid substitutions.11. The controlled-release pharmaceutical formulation according to anyone of claims 7 to 10, wherein said polymer is an acid-terminatedpolymer.
 12. A controlled-release pharmaceutical formulation comprisinga modified bioactive agent encapsulated by a polymer, wherein theisoelectric point of said modified bioactive agent has been decreasedrelative to a parent molecule to reduce the initial diffusion rate ofthe agent from the polymer.
 13. The controlled-release pharmaceuticalformulation according to claim 12, wherein the modified bioactive agentcomprises additional negative charge relative to a parent molecule. 14.The controlled-release pharmaceutical formulation according to claim 13,wherein said modified bioactive agent is conjugated to anegatively-charged accessory molecule.
 15. The controlled-releasepharmaceutical formulation according to claim 13, wherein said modifiedbioactive agent comprises negatively-charged amino acid substitutions.16. The controlled-release pharmaceutical formulation according to anyone of claims 12 to 15, wherein said polymer is an ester-terminatedpolymer.
 17. A controlled-release pharmaceutical formulation comprisinga modified bioactive agent encapsulated by a polymer, wherein theisoelectric point of said modified bioactive agent has been increasedrelative to a parent molecule to increase the initial diffusion rate ofthe agent from the polymer.
 18. The controlled-release pharmaceuticalformulation according to claim 17, wherein the modified bioactive agentcomprises additional positive charge relative to a parent molecule. 19.The controlled-release pharmaceutical formulation according to claim 18,wherein said modified bioactive agent comprises a positively-chargedaccessory molecule.
 20. The controlled-release pharmaceuticalformulation according to claim 18, wherein said modified bioactive agentcomprises positively-charged amino acid substitutions.
 21. Thecontrolled-release pharmaceutical formulation according to claim 18,wherein said modified bioactive agent further comprises an acetatecounter ion.
 22. The controlled-release pharmaceutical formulationaccording to any one of claims 17 to 21, wherein said polymer is anester-terminated polymer.
 23. The controlled-release pharmaceuticalformulation according to any one of claims 1 to 6, and 12 to 22, whereinsaid polymer is a non-biodegradable polymer.
 24. The controlled-releasepharmaceutical formulation according to any one of claims 1 to 6, and 12to 22, wherein said polymer is a biodegradable polymer.
 25. Thecontrolled-release pharmaceutical formulation according to any one ofclaims 2 to 4, 8 to 11, and 18 to 22, wherein said additional positivecharge is distributed uniformly across the bioactive agent.
 26. Thecontrolled-release pharmaceutical formulation according to any one ofclaims 2 to 4, 8 to 11, and 18 to 22, wherein said additional positivecharge is distributed non-uniformly across the bioactive agent.
 27. Thecontrolled-release pharmaceutical formulation according to any one ofclaims 13-16, wherein said additional negative charge is distributeduniformly across the bioactive agent.
 28. The controlled-releasepharmaceutical formulation according to any one of claims 13-16, whereinsaid additional negative charge is distributed non-uniformly across thebioactive agent.
 29. The controlled-release pharmaceutical formulationaccording to any one of claims 1 to 28, wherein said bioactive agent isa peptide molecule.
 30. The controlled-release pharmaceuticalformulation according to any one of claims 1 to 29, wherein saidcontrolled-release pharmaceutical formulation is a microparticle.
 31. Amethod for increasing bioactive agent loading efficiency in apolymer-based delivery system, comprising modifying the isoelectricpoint of the bioactive agent prior to encapsulation in the polymer-baseddelivery system.
 32. The method according to claim 31, wherein theisoeletric point of the bioactive agent is increased such that itcarries a more positive charge in the environment of the polymer-baseddelivery system.
 33. A method for modulating the erosional release rateof a bioactive agent from a polymer-based delivery system, comprisingmodifying the isoelectric point of the agent prior to encapsulation inthe polymer-based delivery system.
 34. The method according to claim 33,wherein the erosional release rate is increased by quantitativelyincreasing or decreasing the isoelectric point of the bioactive agentsuch that it carries a greater net positive or negative charge,respectively, compared to the parent molecule in the environment of thepolymer-based delivery system.
 35. The method according to claim 34,wherein the isoelectric point of the bioactive agent is increased ordecreased by adding additional positive or negative charge to a parentmolecule to produce a stoichiometric increase or decrease in net chargerelative to the parent molecule.
 36. The method according to claim 35,wherein additional positive charge is added to a neutral or cationicparent molecule to increase the erosional release rate.
 37. The methodaccording to claim 35, wherein additional negative charge is added to aneutral or anionic parent molecule to increase the erosional releaserate.
 38. A method for modulating the initial diffusional release of abioactive agent from a polymer-based delivery system, comprisingmodifying the isoelectric point of the agent prior to encapsulation inthe polymer-based delivery system.
 39. The method according to claim 38,wherein the isoelectric point of the bioactive agent is increased ordecreased such that it carries a greater net positive or negativecharge, respectively, relative to the parent molecule in the environmentof the desired polymer-based delivery system.
 40. The method accordingto claim 39, wherein the initial diffusional release rate is increasedby adding additional positive charge to the parent molecule to produce astoichiometric increase in net charge relative to the parent molecule.